Abstract
Background ; We here present a first intraspecific eco-variant
post natal attempt at evaluation of skull signaling in the small African
pangolin (Boreoeutheria) family, sub-family phatagininae emphasizing
evolutionary trend in ontogeny. Forty; digitally processed skulls and
foramen magnum outlines of this species from different geo-locations
were assessed for asymmetry, foramen magnum shape variations and
modularity hypotheses on skull areas using geometric and Elliptical
Fourier analyses methods. Results ; Regression of log
transformed centroid size and asymmetric components suggested
significant expression of asymmetry through ontogeny (p˂0.03) with size
accounting for 81.34% of asymmetric shape changes. Multivariate
analysis of regression confirmed directional but low (p˂0.5) fluctuating
asymmetry (F1539=3.4045, F882= 3.2665,
for dorsal and ventral views respectively). Intraspecific ontogenetic
allometric trajectories followed rostro-caudal and caudo-lateral
directions; intercepts for shape/size predictions were parallel.
Mahalanobis distances between centroids (2.42) of specimens were
significant (p˂ 0.01). Variance-covariance matrix in ontogeny lies
between 0.0017 and 0.56 suggestive of shape overlaps and variations.
Foramen magnum outline descriptors by incremental harmonics explained
morphologic details; the first 4 effective principal components defined
96.98% of shape properties, while (3.02%) constituted finer details.
74.1% accuracy decline after size factor elimination. Modulation PCA:
of Covariance Matrix and Asymmetry component was 88.38% and 7.48% (PC1
and 2) variance % predicted 10.08%. Conclusions ; The study
confirmed directional ‘handedness’ and fluctuating asymmetries among
skull samples studied, an attenuated ability to maintain paired
symmetric bilaterality irrespective of geo-location. Foramen magnum
shape assumed priority over size in ontogeny with profound asymmetry
(from the 5th harmonic); an indication of precocity
and early presumptive form for arboreal adaptation this observation in
conjunction with modularity inference suggested instability and global
weakness of cranial integration processes and modules. These findings
will be of value for species conservation and may be vital in pangolin
evolutionary systematics.
Keywords- African Small Pangolins, Allometry, Foramen magnum,
Asymmetry, Skull modularity, Geometric morphometrics
Background
The small African pangolin (SAP) is a poorly described scaly anteater
formerly classified as a single family Manidae from the Order Pholidota
(Murphy et al., 2001a). Recent research has reclassified those (P.
tricuspis ) to the Clades of Boreoeutheria, Sub-order Ferungulata,
sub-family Phatagininae (Gaubert et al., 2017; IUCN, 2018) and are not
related to the giant anteater (Myrmecophaga tridactyla
vermilingua ) of the Order Pilosa from Central South American nativity
whose familiarity to sloths is well documented. Pangolins are better
related to carnivorans in evolutionary trends (Gaudin and Wible, 1999;
Murphy et al., 2001b). All eight extant species of SAP are currently
classified as threatened, three of these are critically endangered
(IUCN, 2018). SAP are arboreal in nature with Sub-Saharan African
geographic distribution (ranging from West, Central to Southern Africa),
they are deeply diverged from the Asian and Indian species of the genusManis (Gaubert et al., 2017; Gaudin et al., 2019). Several
morphological variations of its head and cranium have brought this
species to fore in current researches and further tilted her already
threatened status to critical level (Ferreira-Cardoso et al., 2020);
this new status justified this study more so that morphological
distinction of species is a requisite vulnerability evaluation in the
instance of ecology and evolutionary status classification. The species
has been declared the most trafficked in the world (Gaubert et al.,
2017); this fact serves a major impetus to study its peculiar cranial
morphology as well as certain developmental/ biological processes as an
adjunct to molecular tracing of trafficked mammals for preservation and
conservation. Tracking and tracing smuggled species skeletons and
remains through illegal trade routes often require adequate information
on geographic distribution, shape and sizes of preserved antiquated or
archival specimen as well as season of interception to offer effective
control and discourage poaching.
Attempts at preservation and conservation of critically threatened
species such as the small African Pangolins (Phataginus tricupis )
have been made through extensive captive breeding; this is partly due to
shrinking ecology and urbanization and have been documented to interfere
negatively with natural selection (Hewitt, 2001); a phenomenon
associated with founder’s events as well as increased occurrence of
developmental aberrations in species (Hewitt, 2001), hybridization, is
another common occurrence when wild-taken variants are introduced in
breeding programs. Preservation effort may only be successful when
baseline information on proper characterization and developmental
peculiarities of species structural parts is possible (Richtsmeier and
Deleon, 2009).
The zygomatic, maxillary and temporal bones of the skull developed from
the first pharyngeal arch (Elliot, 2010) whereas the neural crest andmesenchymal cells forms the cranium (Le Lievre, 1980; Le Douarin
and Kalcheim, 1999). Modularity hypotheses of development will be tested
using landmark partitions to verify if co-variations among embryological
related landmarks are lower in comparison to partitions based on
anatomical proximities. A combination of three separate embryological
compartments will be analyzed for dependency for all partitions in both
planes.
Asymmetry as a concept may be defined by the distribution of
left-to-right differences in a population (Palmer and Stroebeck, 1986).
Fluctuating asymmetry is relevant in the assessment of environmental and
developmental stress in organisms. Subtle and unapparent directional
asymmetry (DA) has been described fairly regularly among species
population using the methods of geometric morphometrics and this seems
to be fairly widespread (Klingenberg et al., 1998). The present
investigation is to the better of our knowledge a first post natal
developmental investigation on the skull of the small African Pangolin.
Calvarias development aims at achieving a symmetric paired structure
(Elliot, 2010), deviations from this ’target’ morphology could result
from individual or population developmental stress/noise (ecologic
input) (Urbanova et al., 2014) and can be useful in making inferences
about such biological processes since morphological asymmetry may
provide information on a variety of biological mechanisms (Polak, 2003).
There is lack of documented evidence on the development of the skull in
this species, thus making accurate taxonomic classification and
discrimination among similar extant species conjectural especially for
trafficked Pangolin. The challenge of skeletal remains recovery and
failure of formulated conservation policies resulting from wrong
identification created a knowledge gap to be bridged by this study.
Visual observation of sample remains of various ages revealed minute
subtle skull asymmetries despite the absence of masticatory mechanisms
prompting such queries to warrant enquiry into the extent and certainty
of developmental errors to signify the existence of developmental
instability, poor integration and a suspicion of reduced ability to
maintain bilaterality in paired symmetric skull structures taken from an
environment with ecologic constraints, an observation relatively scarce
in arboreal species.
Significant overlap of body structural similarities and divergence
between P. tricuspis and all other extant species and betweenP. tetradactyla and P. javanicus respectively have been
reported by Ferreira-Cardoso et al. (2020), a situation which provokes a
closer investigation on its (SAP) skull characterization.
Latest available literary evidence on extant Pangolins; Manidae
(Ferreira-Cardoso et al., 2020) focused on comparative skull shape
variations but was not contextual with phenotype as a product of
environmental, ecologic and evolutionary trends in the order Pholidota.
The following are the aims and objectives of this study:
- This study aims to investigate the association or otherwise between
environment/ecology and skull development in SAP to justify observed
skull asymmetries.
- That the possibility of ‘handedness’ in skull side’s use and
development despite absence of teeth for mastication does not exist in
the sample population evaluated (null hypothesis), particularly due to
paucity of documented evidence of directional asymmetric
investigations in the species from different ecological background
under study.
- Establish and compare variations among sample populations in foramen
magnum outline allometry and possible occurrences of malformations
such as dorsal and ventral notches among population for an assessment
of stability in this structure.
- Assess overall evaluated skull developmental pattern among contiguous
landmarks (modularity hypothesis) to confirm or otherwise an
association in embryonic skull modules compared to related anatomic
structures in its integration process using two blocks Partial least
squares (PLS), Overall strength of association between blocks: yielded
lowest RV coefficients; Permutation test against the null hypothesis
of independence with 10,000 rounds of randomization.
. In evaluation of symmetry/asymmetry in skull construction and
architecture, all contributory forms of asymmetry (directional
asymmetry, fluctuating asymmetry and antisymmetry) were explored (Palmer
and Strocker, 1986). We also aimed at studying the foramen magnum
construction in ontogeny from the three ecologies as a corroborative
attempt in characterization of observed asymmetry of this structure for
confirmation of in-born errors. Further, we endeavor an evaluation of
ontogenetic morphological features using a range of harmonics in
Elliptical Fourier Analysis in resolution of foramen magnum outlines
architecture.
0. Materials and Methods
ETHICAL CONSIDERATIONS
Ethical approval for the use of these animals was granted by the Animal
Care and Use, Research Ethics Committee (ACUREC), University of Ibadan,
Nigeria, ethical code number UIACUREC/17/0023.
ANIMAL ACQUISITION, SKULL PROCESSING AND CATEGORIZATION
To enhance validity, samples for this study were taken from separate
locations in Nigeria. All skulls were obtained after due permissions
from museums curators of Universities in Nigeria; Eighteen (18), twelve
(12) and six (6) from the Department of Anatomy, University of Ilorin,
Kwara State, Department of Veterinary Anatomy, Federal University of
Agriculture Makurdi, Benue State, and Department of Veterinary Anatomy,
University of Ibadan, Oyo State, respectively. Four (4) unsexed were
wild taken in Ibadan, Oyo State, categorization was based on skull size
and geographical location (Fig. 1) while animal handling was in
conformity with ACUREC and NIH guidelines for Use and Care of Laboratory
Species. Sacrifice and maceration process were according to Catania et
al., (2000) and Igado (2017).
Figure 1 Distribution areas (starred) of the small African
Pangolin (Phataginus tricuspis ) along the coast line in the
tropical rain forest zone with special concentration in the Southwestern
parts of Nigeria which serves as our assessment areas
A series of eleven (11) landmarks comprising of four (4) single midline
(axis of symmetry) and seven (7) paired points on either side of the
skulls were digitally placed on direct dorsal and ventral views (nine
landmarks) of skull images (Fig.2a) using TPS Dig2 Vers. 1.40 (Rholf,
2015) CANON EFS-1200D Camera with EF-S 18-55 IS 11 kit and HAMA tripod
plumb line stand stabilizers at a focal distance of 2.8 cm and 15 cm
DIN, shutter speed ISO 1/100. The study utilized Generalized Procrustes
Analysis (GPA), which superimposed landmark configurations, apart from
providing information on size (Centroid size, CS, calculated as the
square root of the sum of squared distances of each landmark from the
centroid of the landmark configuration) and shape (Procrustes distance
estimates) eliminates spatial variation that does not correspond to
form. Allometric data could therefore be assessed for evaluation as
analysis of size and shape differentiation across the geo-ecologies.
Sage: software was used in the evaluation of asymmetric and symmetric
components for this study, Symmetry and Asymmetry in Geometric Data
Version 1.21 software (Marquez, 2014) freely available online at
http://www-personal.umich.edu/~emarquez/morph/